The dopamine (DA) transporter (DAT) is a membrane protein that regulates dopaminergic neurotransmission by mediating uptake and reverse transport (release) of DA in a Na+-dependent manner. We wish to elucidate the molecular details governing binding and translocation of substances targeting DAT as a first step in the development of therapeutic interventions for diseases involving DAT, such as psychostimulant (cocaine and amphetamine) dependence, neurodegeneration, and psychiatric disorders. The specific aims in this proposal are: 1) Characterizing the relationship between DAT activity and the intracellular Na+ level. The hypothesis is that the intracellular Na+ level under depolarizing conditions and thus the action of Na+ channels regulates DAT activity. We will correlate the Na" level inside cells expressing both DAT and voltage-gated Na+ channel with the ability of DAT to bind and transport DA;and we will probe conformational changes induced by intracellular Na+. 2) Delineating sidedness for action of various substrates at DAT. The hypothesis is that substrates with a modified catechol moiety differ from DA in their ability to access the binding site from the intracellular side of DAT. We will use different DAT preparations, chemical modification, and functional assays to investigate which side of the plasma membrane action of substrates is initiated from;and we will combine structure-activity studies with site-directed mutagenesis to explore, for both substrate and DAT, the structural determinants involved in external and internal access. 3) Assessing functional role of DAT oligomerization. The hypothesis is that oligomeric structures of DAT play a role in asymmetric binding and translocation of substrates, binding of cocaine analogs, conformational changes, and substrate-induced DAT internalization. We will use cells co-expressing two different DAT proteins to determine the minimal function unit for binding and transport, to explore the contribution of inter-monomer interactions to conformational changes of DAT, and to address the relation of oligomerization to the substrate-induced DAT internalization. The planned studies will provide information on the function of DAT in relation to protein structure as well as on the molecular mechanisms of substrate-type psychostimulants or parkinsonism-inducing toxins, and may lead to identification of asymmetrically-acting substrates that could be tools for DAT studies. Results may also help understanding the closely related serotonin and norepinephrine transporters that are linked to mental illness.